Microscope turret mounted laser epi-illumination port and method for calculating and displaying the isothermal contours produced by a laser

ABSTRACT

An optical injection system for use in conjunction with a microscope having a turret supporting an objective having an optical path is provided. The optical injection system includes a dichroic mirror disposed between the turret and the objective, along the optical path. A collimating lens is further provided having an optical path directed to intersect the dichroic mirror. A laser source is positioned to project a laser beam through the collimating lens and along the collimating lens optical path. The laser can be used in conjunction with the microscope for a number of microsurgery applications. A method for calculating and displaying the isothermal contours of the energy produced by a laser in a sample is also provided.

RELATED APPLICATION

[0001] This application claims priority to co-pending U.S. ProvisionalApplication No. 60/329,769, filed on Oct. 16, 2001, and co-pending U.S.Provisional Application No. 60/366,774, filed on Mar. 22, 2002,incorporated herein in their entirety by this reference.

FIELD OF THE INVENTION

[0002] The present invention relates to an adaptation of a laser to amicroscope. More specifically, a device is disclosed that fits betweenthe microscope objective and the microscope nosepiece turret forintroducing laser energy into the optical path and on to the microscopesample.

[0003] The present invention also relates to a method for calculatingand displaying the isothermal contours produced by a laser in a sample.The method can be carried out using the described microscope and acomputing apparatus for performing the calculations and displaying theisothermal contours.

BACKGROUND OF THE INVENTION

[0004] Recent advances in biology and medicine have led to thedevelopment of laser beam microsurgery on cells. The laser beam is welladapted to micro-manipulation of small objects, such as single cells ororganelles. It provides the advantage of non-contact ablation,volatilization, sterilization and denaturing, cutting, and other formsof thermal and actinic-light treatment. The four parameters of focalspot size, laser wavelength, pulse duration, and laser power, provide avariety of regimes suitable for different applications. One example useof laser beam microsurgery is the application of laser beams to thetreatment of the mammalian oocyte and embryo. However, laser beammicrosurgery in a number of inverted or upright microscopes can beutilized for many different surgical or medical applications.

[0005] In further detail of the example application, the early-stagemammalian embryo is contained within a protective layer, the zonapellucida (“ZP”). The ZP is relatively analogous to the shell of a hen'segg. This proteinaceous ZP layer is of varying thickness, typically 10to 20 μm, and of varying hardness. The embryo remains within the ZPduring development from the single-cell to the blastocyst stage, atwhich point the embryo breaks out of the ZP and implants itself into theuterine wall.

[0006] It has been found that certain embryos, typically those fromolder mothers or embryos that have been frozen for storage, frequentlyhave much tougher ZP layers than younger-origin or untreated embryos.Consequently, when the time comes for the embryo to emerge from the ZP,there may be a significant impediment in the tougher layers, which haveto be traversed. If the embryo fails to hatch in the limited timeavailable, it will be lost and fertility will fail.

[0007] Assisted hatching derives from the observation that fertility canbe augmented by generating holes or gaps in the ZP through which theembryo can more easily emerge. This has been done using mechanical orultrasonic cutting, chemical erosion (acidified Tyrodes solution), or bylaser ablation of part of the ZP.

[0008] A laser can be used to produce a trench in the edge of the ZPlayer, penetrating through (or almost through) the ZP thickness. Thetrench creates a weakened region that provides a crack through which theembryo will later emerge. Lasers of many types can ablate the ZP. Alaser of wavelength λ=1480 nm, which is strongly absorbed in water, hasbeen found to be effective for thermolysing the ZP. The laser can beused in pulses relatively short enough to avoid significant thermalconduction into the nearby embryo blastomeres, and at the same timeavoid a chemical effect on the cellular chemistry, since it is in thenon-actinic infrared wavelength region. The ZP is removed out to aradius around the laser beam determined by the local temperature historyduring the laser pulse.

[0009] The same laser system can be used to ablate a larger region of ZPso that an intact blastomere can be removed for external analysis. Thelaser is used, in this case, in a series of pulses directed at adjacentparts of the ZP to erode away a larger region. Typically, a gap isopened until a pipette can be introduced to suck out a blastomere. Theembryo is relatively resilient, and generally survives both hatching andbiopsy ZP ablation.

[0010] Another related application of the laser system is in directremoval of the polar body for genetic analysis. Removal can be doneeither at the oocyte stage (first polar body), or after fertilization atthe embryo stage (second polar body). Both polar bodies can be used toderive information on the genetic composition of the embryo. Theprocedure is analogous to laser assisted biopsy, except that in thiscase only the ZP layer and not the perivitelline membrane is penetrated,since the polar body remains between the perivitelline layer and the ZP.Additional related applications include transfer of part or all of thenucleus (nuclear transfer or transgenetic engineering), and ablation anddestruction of part or all of the cell nucleus or oocyte spindle (e.g.in a cell to be used as the recipient of nuclear transfer). All of theseapplications benefit from the precise ablation capabilities of the lasersystem.

[0011] The above applications are by way of example and should not beconstrued as limiting the possible uses of the invention. The presentinvention can be applied in any field where a laser beam is used with amicroscope assembly.

SUMMARY OF THE INVENTION

[0012] There is a need in the art for a configuration for introducinglaser energy to a microscope, wherein the length of the laser beamemitted is reduced, and the quality is improved. There is also a need inthe art for a method of calculating and displaying the isothermalcontours associated with the energy generated in a sample by a laser.The present invention is directed toward further solutions to addressthese needs.

[0013] An optical injection system for use in conjunction with amicroscope having a turret supporting an objective having an opticalpath is provided. The optical injection system includes a dichroicmirror disposed between the turret and the objective, along the opticalpath. A collimating lens is further provided having an optical pathdirected to intersect the dichroic mirror. A laser source is positionedto project a laser beam through the collimating lens and along thecollimating lens optical path.

[0014] In accordance with further embodiments of the present invention,the laser source includes an internal laser powered from an electronicpackage. The laser source can further originate using fiber optic cableproviding optical energy from a laser. The laser can be one of aninternal laser and an external laser, and operate at a wavelength (λ) ofabout 1480 nm. The laser beam can also be confocal with an opticalimage.

[0015] The microscope can take the form of an inverted or a non-invertedmicroscope. The microscope objective can take the form of a shortenedoptical train to provide a standard total length 45 mm parfocalobjective unit module. The system can also be adapted to longerparfocality systems, e.g., the Nikon CFI 60 optics (60 mm parfocalitysystem).

[0016] In accordance with another embodiment, the turret has a removableturret adapter. The turret adapter can be one of a plurality of turretadapter designs, thereby creating a universal mounting system, whichenables the optical injection system to be mounted to the microscope.

[0017] In accordance with further embodiments of the present invention,an electronic package can be disposed separate from the system.Alternatively, the electronic package can be built-in to a board in acomputing apparatus. The laser can be mounted on a piston in a cylinder.Focus of the laser in such an arrangement can be provided by a singlescrew to adjust the distance from the laser to the collimating lens.

[0018] In accordance with another embodiment of the present invention,the laser may be located in the electronic package, and the laser energytransferred to the optical injection system through a fiber cable.

[0019] The system of the present invention can be utilized in a numberof different applications, including ablating, dissecting, moving,holding, or otherwise effecting biological cells or tissue with a lasersource.

[0020] A method for calculating and displaying the isothermal contoursgenerated by a laser in a sample is also provided. The method includesapplying a laser beam to the focal point of a sample, dividing theregion near the focal point into cylinders coaxial with the beam,deriving the maximum temperature reached during the laser pulse of atleast three points at arbitrary distances from the focal point, plottingthe temperatures calculated as a function of distance from the focalpoint sufficient to generate isothermal contours, and generating acomputer display of said isothermal contours corresponding to thetemperature calculations.

[0021] In accordance with further embodiments of the present invention,the sample is placed in an isotropic medium and the temperaturecalculations are displayed as rings centered around the focal point. Apicture of the sample can also be displayed with the rings.

[0022] The method of the present invention can be utilized inconjunction with a number of different applications, including ablating,dissecting, moving, holding, or otherwise effecting biological cells ortissue with a laser source.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The features and advantages of the present invention will becomebetter understood with regard to the following description andaccompanying drawings, wherein:

[0024]FIG. 1A is a diagrammatic illustration of a conventionalfluorescent port laser;

[0025]FIG. 1B is a diagrammatic illustration of a conventionalfilter-cube laser;

[0026]FIG. 1C is a diagrammatic illustration of a conventionalfilter-cube fiber optic laser;

[0027]FIG. 2A is a side view illustration of a laser and microscopearrangement according to one aspect of the present invention;

[0028]FIG. 2B is a side view illustration of the arrangement of FIG. 2Ain further detail according to one aspect of the present invention;

[0029]FIGS. 3A and 3B are perspective illustrations of a fiber optic andmicroscope arrangement according to one aspect of the present invention;

[0030]FIG. 4 is an exploded diagrammatic illustration of a firstembodiment of the laser assembly according to one aspect of the presentinvention;

[0031]FIG. 5 is a flowchart illustrating the capture of images andconveyance to the computing apparatus according to one aspect of thepresent invention;

[0032]FIG. 6 is a flowchart illustrating the generation of isothermalrings according to one aspect of the present invention;

[0033]FIG. 7 shows the isothermal contours generated by applying a laserpulse of power 140 mW and duration 3 msec to the ZP of a pre-embryoniccell. The central ring shows corresponds to the laser Gaussian beamdiameter, and the rings successively radiating from the central ringshow the peak temperatures of 140, 100, 80, 60 and 50° C., respectively.

[0034]FIG. 8 shows laser ablation of ZP of bovine eggs at 100 mW, pulseduration 1.5, 3, 4.5 ms.

[0035]FIG. 9A shows bovine eggs in which several channels have been cutby laser.

[0036]FIG. 9B shows a close-up view of the channels in bovine ZP. Thechannels are approximately 25 μm long, almost constant-radiuscylindrical intercepts with sharp edges and do not show effects of beamconvergence or divergence.

[0037]FIG. 10A shows FEA analysis of 3 ms pulse, 100 mW, convergingGaussian attenuated beam solved in half plane. The isotherms at 0.1 msinto the pulse are indicated. Isotherm interval is 2.5°. Centraltemperature is 98.8° C.

[0038]FIG. 10B shows FEA analysis of same case as in FIG. 9A, at 3 ms.Isotherm interval is 2.5° C. Central temperature is 189° C.

[0039]FIG. 11 shows GE analysis of flattop standard case (100 mW, 3 ms,a-3 μm), beam on axis of a 200 μm right cylinder. Temperature given atvarious radial distances on focal plane. Using a Gaussian-profile beamincreases the central temperature peak by about 15° C., but has anegligible effect on the temperature exterior to the beam.

[0040]FIG. 12 shows the measurement of beam steering (horizontal view).

[0041]FIG. 13 shows the bending of ray with impact factor b near acentral refractive field (vertical view).

[0042]FIG. 14 shows the probe beam steering angle in the IR laserthermal field versus delivered IR power. Measurements are denoted bysymbols and predictions by solid lines. The dashed line corresponds tothe prediction when maximum temperature cannot exceed 100° C.

[0043]FIG. 15 shows the measured channel diameter versus pulse durationaveraged over three bovine eggs and two murine embryos. Predictedmaximum temperature in focal plane at given radius is shown versus pulseduration and radial distance from axis.

[0044]FIG. 16 shows the predicted maximum of the temperature pulse 20 μmfrom beam axis in the focal plane is given versus laser pulse durationand beam power. Also shown are limit lines corresponding to beam centraltemperature 150 and 110° C.

DETAILED DESCRIPTION

[0045] An illustrative embodiment of the present invention relates to asystem combining a laser with a microscope to result in an efficient andmulti-functional device for performing micromanipulation of smallobjects with a laser.

[0046]FIGS. 2A through 16 illustrate example embodiments of a laserassembly and/or a laser and microscope arrangement, in addition toexample applications for use of the laser and microscope arrangement,and results thereof, according to aspects of the present invention.Although the present invention will be described with reference to theexample embodiments illustrated in the figures, it should be understoodthat many alternative forms can embody the present invention. One ofordinary skill in the art will additionally appreciate different ways toalter the parameters of the embodiments disclosed, such as the size,shape, or type of elements or materials, in a manner still in keepingwith the spirit and scope of the present invention.

[0047] Lasers have been utilized for manipulation of cells and cellularorganelles. The application of a precise dose of radiation for a settime in a pre-designated area can be used to destroy or neutralizeorganelles, such as the nucleus, as well as cutting, trapping andheating entire cells. The present disclosure utilizes an example ofusing the lasers for drilling into embryo zona pellucida (“ZP”).However, this is merely an illustrative application and in no way is itintended that the present invention be limited to such application.

[0048] Adapters and kits used in conjunction with microscopes are shownin two patents described below. U.S. Pat. No. 5,349,468 is directed toan adapter for a microscope that fits between the objective and themicroscope turret for providing fluorescence microscopy. In this devicea light source provides illumination in a direction normal to the opticaxis, which projects the light after collimation onto a dichroic mirror,and then to the objective, to give epifluorescent illumination of thetarget.

[0049] U.S. Pat. No. 4,884,880 is directed to a “kit” that converts thestandard microscope into a single aperture confocal scanningepi-illumination microscope. The kit fits between the objective and themicroscope for the purpose of creating scanning epi-illumination.

[0050] In a conventional fluorescent port microscope arrangement, laserradiation is conveyed from the source to the microscope objective usinga collimated beam and an on-axis dichroic mirror (see FIG. 1A). Theoptical system is analogous to an epifluorescent design, in which laserradiation is used in place of the exciting fluorescent illumination.Chromatic aberration between the laser infrared (IR) beam and visiblelight is removed by adjusting the degree of collimation of the laserbeam, so that it is confocal with the visible image and produces itsmaximum effect exactly in the same plane as the image. In someconventional devices, long IR pathlengths were used to convey the laserenergy between a laser source 10, generally at the back of themicroscope, through a fluorescent cavity 12, on to a dichroic mirror 14,and through an objective 16 to the target. The objective 16 must behighly transmissive in both the laser radiation wavelength and thevisible wavelength for such an arrangement. The long path lengthresulted in attenuation of the laser beam and reduced its safety, sinceprevention of local blastomere heating is more difficult with lesspowerful beams. A typical arrangement of this type is shown in FIG. 1A.

[0051] More recent conventional designs, as shown in FIG. 1B, minimizethe pathlength between a laser source 20 and an objective 22 bypackaging an entire optical system 24, including a dichroic mirror 26,into a region under the objective 22. This is the same location wherethe fluorescent filter-cubes are placed in fluorescent operation. Thisarrangement increases the laser power available and makes the laser moreportable and easily adjustable. However, the arrangement prevents use offluorescent illumination, or other optical devices needing the samespace (e.g. the SpindleView™ polarizer, made by CRI, Inc. of Woburn,Mass., for detecting the oocyte spindle for ICSI), and the laser packagegenerally must be removed before devices of such type can be utilized.

[0052] In certain other conventional designs, the IR radiation isconveyed to an objective 30 using a fiber-optic cable 32 as shown inFIG. 1C. The exit of the fiber-optic cable 32 acts as a laser source,and the beam passes through a collimating lens 34, which may be, forexample, an aspheric or a GRIN lens, and reflects from a dichroic mirror36 to provide the required almost-parallel beam into the objective 30.This arrangement has the advantage of facilitating the positioning ofthe fiber-optic “source”. Moreover, another (e.g. a visible) laser cablecan be spliced into the fiber-optic cable 32, giving in effect twocoincident sources. However, the transfer of energy into and out of thefiber optic cable inevitably reduces the laser power available, due tofocusing, reflective, and cable attenuation losses. The available energytherefore tends to be lower in fiber optic systems, again leading to theuse of longer pulses and the associated greater heating of neighboringblastomeres.

[0053] One difficulty associated with laser power delivery by fiberoptic cables is that, due to the very small diameter of the fiber, thepresence of tiny particles or other obscuration at the cable entry orexit can greatly diminish the intensity of the laser beam delivered tothe target. While this is a common issue for the conventional digitalinformation transfer application of fiber optic cables, such digitalsignals can sustain a loss and still transmit information properly.However, it is a greater potential difficulty with energy-transferapplications where the unreliability of the beam intensity at targetmakes accurate and repeatable ZP hole-drilling problematic.

[0054] The disadvantages of the prior methods can be overcome by theteachings of the present invention. In accordance with the presentinvention, advantage is taken of the fact that a laser source ispackaged in a small integrated-circuit “can” 66 shown in FIG. 2B, and issufficiently small that it may be positioned very close to an objective54. In one embodiment of the present invention, a laser assembly 76including a specially designed low-profile objective (e.g. a short 21 mmobjective) is combined with an optical injection system 56 to introducethe laser light between a microscope turret 58 and the objective 54. Theturret 58 (see FIG. 2A) includes a removable turret adapter 59. Theturret adapter 59 is one of a number of different turret adapterdesigns. The plurality of turret adapter designs creates a universalmounting system, which enables the laser assembly 76 to be mounted toany microscope.

[0055] In this design, a fluorescent filter-cube channel 60 (see FIG.2A) beneath the inverted microscope turret 58 is left unoccupied, andmay be filled with any optical equipment required, since it is no longerintegral to the optical design of the laser system. The entire laserassembly 76 exists between the microscope turret 58 and a microscopestage 62 as shown in FIG. 2A. The position of the laser assembly 76between turret 58 and stage 62 is indicated.

[0056] The optical injection system 56 is shown in additional detail inFIG. 2B. A solid-state laser beam is directed through an adjustablecollimating lens 68 on to a dichroic mirror 70. The dichroic mirror 70reflects the wavelength λ=1480 nm radiation beam along the optic axisand up into the objective 54, in a direction opposite to the image beamlight. A laser diode 66 is mounted inside a piston 72 of precisedimensions, which slides in a cylinder 74. The sliding movement of thepiston 72 controls the distance of the laser diode 66 to the collimatinglens 68, while preventing lateral movement. Any chromatic aberrationbetween the laser beam and the visible image is removed by adjusting thepiston 72 position. The movement of the piston 72 focuses the laserbeam.

[0057] The laser diode 66, piston 72, collimating lens 68 and dichroicmirror 70 are packaged in optical injection system 56. The objective 54screws in to the top of the optical injection system 56, creating thelaser assembly 76, which can be coupled to the turret 58 by way of theturret adapter 59. The entire laser assembly 76 is constructed so thatit can be mounted like an objective, by first screwing the turretadapter 59 on to the objective nosepiece turret 58 and then screwing thelaser assembly 76 on to the turret adapter 59. The design of the laserassembly 76 to turret adapter 59 interface is such that the laserassembly 76 may be oriented in any rotational position and then lockeddown into place. This feature overcomes the problem of a fixed threaddesign, which would have only one final rotational position. In theexample embodiment, the total length of the laser assembly 76 includingthe length of the specially designed objective 54 is equal to the lengthof a standard 45 mm parfocal objective. The complete laser assembly 76fits between the turret 58 and the stage 62 of a microscope. Althoughthe system is designed for use in inverted microscopes, it can easily beused in upright microscopes as well.

[0058] In the particular application of ZP hatching and embryo biopsy, ashortened objective 54 is utilized, with magnification 50× and NA=0.6.The objective 54 is made to transmit more than 90% power at thewavelength of λ=1480 nm. The laser diode 66 is precisely positionedwithin the laser assembly 76, and is maintained in the required rigidoptical relationship with the other parts of the laser assembly 76. Theoptical injection system 56 therefore remains with the required focaland directional alignment between visible and infrared beams. The entirelaser assembly 76 can therefore be transferred to other microscopes 65(see FIG. 2A) without changing the relationship between the opticalcomponents forming the laser assembly 76, and without requiringrealignment.

[0059] Electrical power to the laser diode 66 is provided from astandard control circuit 78 on a conventional printed circuit board(PCB) 80. The PCB 80 may be mounted in a separate box, or as a boardinside a computing apparatus 82. A cable 84 between the computingapparatus 82 and the laser assembly 76 connects each component to theother. The cable 84 normally connects to the laser assembly 76 with arotational swivel, such that the microscope turret 58 (see FIG. 2A), andany of the other objectives used, can rotate without causinginconvenience from the controlling cable 84. It should be noted thatinclusion of Zener and Schottky protection diodes (not shown) mounted oninterface PCB 106 of FIG. 4 within the laser assembly 76 minimizes riskof standard electrostatic-damage to the laser diode 66. The laser diode66 is further protected by automatically shunting the laser diode 66connections when the control cable 84 disconnects from the interface PCB106 within the laser assembly 76.

[0060] A software application installed on the computing apparatus 82controls the laser assembly 76 (laser power, laser pulse length, lasermultipulse). The phrase “computing apparatus” as used herein refers to aprogrammable device that responds to a specific set of instructions in awell-defined manner and can execute a set of instructions. The computingapparatus 82 can include one or more of a storage device, which enablesthe computing apparatus to store, at least temporarily, data,information, and programs (e.g., RAM or ROM); a mass storage device forsubstantially permanently storing data, information, and programs (e.g.,disk drive or tape drive); an input device through which data andinstructions enter the computing apparatus (e.g., keyboard, mouse, footswitch, or stylus); an output device to display or produce results ofcomputing actions (e.g., display screen, printer, or infrared, serial,or digital port); and a central processing unit including a processorfor executing the specific set of instructions. The computing apparatushas access to code for transmission of the microscope image on to amonitor (not shown). The code includes routines for measuring the image,storing and retrieving electronic images, operating the laser assembly76, and generating and printing reports.

[0061] As shown in FIGS. 3A and 3B a fiber-optic cable 86 may besubstituted for the laser diode 66 in an optical injection system 57. Alaser 77 is then located at the other end of the fiber optic cable 86,together with an electronic laser control circuit 79. This may be builtin to an optical injection system 57, in a separate system, or built onto a PCB for insertion into a computing apparatus. In the latter case,the fiber-optic cable 86 plugs directly into the PCB in the computingapparatus and no electric connections are required between the assemblyand the computing apparatus. In the example embodiment, the laser 77provides the necessary signal through the fiber-optic cable 86 to thedichroic mirror 70, which reflects the signal to the objective 54 asbefore. The substantial difference in this embodiment is the use of thefiber optic cable 86, which can allow the laser 77 source to be locatedin a number of locations proximal or distal from the optical injectionsystem 57.

[0062] The use of fibers has the advantage of producing an almostGaussian beam, allowing relatively good focusing properties. Fibers maybe spliced to introduce a visible beam (for aim spot or focus spot)co-directional with the IR beam. However, fibers also have the sameproblem of particle obscuration mentioned above.

[0063] It should be emphasized that the design used in the presentinvention can be applied to substantially any embodiment of a laser tobe used for microscope irradiation. Some examples include laser cutting,e.g., at a wavelength of λ=337-390 nm, laser scissors and lasertweezers, and laser differential heating. DNA denaturing (e.g. nucleusablation) is another application. The system can be extended to anylaser from which the power is delivered in a fiber-optic cable. Thepresent invention is not limited to inverted microscopes. It can be usedin an upright microscope, and requires no adjustments when transferredbetween one type of microscope and another.

[0064]FIG. 4 illustrates another detailed example embodiment of anoptical injection system 88 built in accordance with the teachings ofthe present invention. A first embodiment of the optical injectionsystem 88 was designed and machined, and is shown in FIG. 4. A body 90houses the elements of the optical injection system 88. A mirror mount92 provides support for a dichroic mirror 102. An adapter nut 94attached to the body 90 with a retainer ring 114 mounts the laser systemto a turret adapter 96. A diode mount 98 and diode clamp 100 support alaser diode 120, while a compression spring 116 pushes against the diodemount 98. A focus screw 104 is used to adjust the laser light. Aconnector on interface PCB 106 is suitable for connecting the assemblywith a computing apparatus (such as the computing apparatus 82 of FIG.2B). A lens bushing 108 positions a lens 118. A cover 110 surrounds thelaser portion of the optical injection system 88. A compact objective112 mounts substantially orthogonal to the laser diode 120 direction.

[0065] The optical injection system 88 was used with a speciallydesigned HTB short lens, and the assembled unit with lens had aparfocallength of 45 mm, equivalent to the length of a standardobjective. The optical injection system 88 was demonstrated on a NikonTE 300 microscope, then transferred to a Leica DMIL, Zeiss Axiovert 25and an Olyrnpus IX-70 microscope. In all cases, the focus of the IR beamdid not have to be adjusted, and the unit gave high quality images.Standard tests on the HTR 3 μm grid showed no detectable imagedifference between the microscopes.

[0066] The IR transmission is also independent of the microscope, sinceit can only depend on the optical injection system 88. The value of thetransmission, T, was measured with the system by removal and replacementof the objective, receiving the beam in an OPHIR Nova NIST-traceabledetector. The result was T=90%, close to the values obtained with thestandard objective.

[0067] The size and quality of the holes drilled in test bovine oocytesamples was measured. The results fell within the predictions expectedfrom the thermal diffusion transfer theory [Douglas-Hamilton DH andConia J (2001)]. This result would be expected since the entire imagingapparatus is contained within the optical injection system 88.

[0068] The teachings of the present invention provide for IR focaladjustment to be required only at installation. The laser assembly canbe fitted to any microscope without changes. The optical injectionsystem works on inverted and on upright direct transmission microscopes.Short optical path gives maximum beam power available. High beam poweravoids damaging local cells that are not targeted. The optical injectionsystem can be adapted to fiber optic sources. The entire electronicsassociated with the optical injection system can be contained insidecomputing apparatus. The optical injection system is small andlightweight, and does not require special installation. The opticalinjection system can further be returned for factory servicing, andre-installed by a user without losing its alignment or requiring opticaladjustments.

[0069] An illustrative embodiment of the present invention also relatesto a method for calculating and displaying the isothermal contoursgenerated by a laser in a sample is also provided. As used herein, theterm “isothermal contours” refers to the array of maximum temperaturesgenerated by a laser pulse at particular locations within a sample,e.g., the ZP of a pre-embryonic cell. The locus of all points reachingthe maximum temperature represents an isothermal contour of maximumtemperature or peak thermal excursion. The method includes applying alaser beam to the focal point of a sample, dividing the region near thefocal point into cylinders coaxial with the beam, deriving the maximumtemperature reached during the laser pulse of at least three points atarbitrary distances from the focal point, plotting the temperaturescalculated as a function of distance from the focal point sufficient togenerate isothermal contours, and generating a computer display of saidisothermal contours corresponding to the temperature calculations.

[0070] The isothermal contours illustrate the thermal effect of a laserbeam on a sample, e.g., the ZP of a pre-embryonic cell. A wavelength ofapproximately 1480 nm radiation is strongly absorbed in water and,therefore, absorbed in all living tissue. Consequently, the region ofthe laser beam in an aqueous medium becomes significantly heated and thetemperature rise at the focal point can easily reach ΔT=150° C. by theend of the laser pulse. The ZP melts at a temperature of about T=140° C.Therefore, the ZP will be removed within a ring corresponding to peaktemperature T of about 140° C.

[0071] The thermal energy deposited by the laser beam is conducted awaythrough the medium. During the laser pulse a dynamic balance is set upbetween the laser heating and the thermal conduction rate which resultsin the temperature rising rapidly until a steady-state temperature isreached. Generally, the pulse is terminated long before thesteady-state. Consequently, regions at different distances from thefocal point reach different temperatures that depend on the pulseduration, the beam intensity, distance from focal point, andconductivity of the medium. The temperature at or near the focal pointof the beam can be as high as ΔT>200° C. The temperature falls rapidlywith increasing distance from the focal point. In order to ensure thesafety of the laser system and the success of the laser beammanipulations, it is very important to accurately calculate theparticular temperatures at various positions of the sample.

[0072] The medium containing the sample should be compatible for varioustypes of organic structures, such as embryos, blastomere, or othercells. These structures will differ in chemical composition from themedium itself and in general will have different thermal properties. Thethermal diffusion equation can be solved in this type of geometry andthe thermal history derived at every point. The locus of all pointsreaching a particular maximum temperature represents an isothermalcontour of peak thermal excursion. However, since the major component ofmost biological structures is water, the samples will have thermal andIR-absorption properties similar to those of water.

[0073] A theoretical derivation of the thermal diffusion regime for anisotropic medium has been given by Douglas-Hamilton and Conia (2001) (J.Biomed Opt. (2001) 6:205-13 “Thermal effects in laser-assistedpre-embryo zona drilling”), incorporated herein by reference. The regionnear the focal point is divided into cylinders coaxial with the beam andthe thermal history at arbitrary distances from the focus is derivedwhich determines the thermal regime or isothermal contour as a functionof distance. Such values also specify the distance from the focusrequired to maintain safety. In particular, these calculations areapplied to cells that are within the embryo while the ZP is beingdrilled, in order to maintain a safe stand-off distance between thedrilling operation and the embryonic cells. The temperature of themedium near the beam was measured experimentally and confirms thetheoretical thermal predictions. Any medium anisotropy can be includedin the calculation and the appropriate isothermal contours derived.

[0074] Using the foregoing thermal profile, the maximum temperaturereached during the laser pulse as a function of distance from the beamaxis is plotted and expressed as a series of isotherms, eachrepresenting the peak temperature reached during (or after) the laserpulse. To first approximation, the medium may be treated as isotropicand the peak-temperature isotherms contours are generated as ringscentered on the beam axis or focal point. The isotherms are displayed ona computer display centered on the beam focal point and can beidentified by color or label to give the local peak temperature computedfrom the thermal diffusion model. Because the local heat input dependson the laser power and the laser pulse duration, the diameter of theisothermal rings will vary as a function of the laser power and pulseduration.

[0075] The computing apparatus 82 can display the isotherms as describedin the above example application, or a different computing apparatus canbe utilized to display the isotherms. Prior to displaying the isotherms,the computing apparatus 82 must first obtain images of the sample asviewed through the microscope. To accomplish this, and as illustrated inFIG. 5, the microscope images are either taken with a digital camera oran analog CCD camera (step 200). If the images are taken with a digitalcamera, or are otherwise in digital format, the images are transferreddirectly onto the monitor screen (step 202). If the images are not indigital format, the analog signal is transferred to a digitizer board(step 204) to be digitized [e.g. the Bandit digitizer/display board fromCoreco Corp, Quebec, or equivalent]. The digitized image is thentransferred directly to the screen (206). In either instance, the imagecan be over-written with information from a digitizing system, so thatany computer information may be superimposed on the image.

[0076] Once the image of the sample is captured from the microscope, thecomputing apparatus 82 takes the image and superimposes a plurality ofisotherms in the form of lines drawn on the image of the sample. Theprocess carried out by the computing apparatus 82 for creating theisotherms can be described as follows, and as illustrated in FIG. 6.

[0077] In the example embodiment, the Isotherms are generated byinterpolating values from fixed solutions for a 180 mWatt beam of radius2 micron. The fixed solutions are computed at pulse durations of 0.5, 1,2, 3, 4, 5, 10, 20 milliseconds (step 210). The isotherms for a beam ofpower 180 mW at any given pulse duration can be accurately interpolatedfrom the fixed solutions (step 212). By way of example, using a thirddegree polynomial interpolation gives results within 5% of the correctvalues. Since the temperature excursion scales directly with beam power,only the computed solutions for different pulse durations at aparticular given beam power are required. The corresponding isothermscan then be computed at any other beam power, for the same beam radius,by direct linear extrapolation (step 214).

[0078] In practice the system is used at maximum power, and the effectof the beam is controlled by decreasing the laser pulse duration. Inthis way, the conduction of heat from the laser beam to local (e.g.blastomere) cells is minimized.

[0079] The corresponding peak temperature isothermal ring diameters arederived from the laser beam power and the laser pulse duration input atthe controls (step 216). For any combination of pulse duration and beampower, there is a single-valued set of rings, corresponding to thepositions at which each peak temperature is reached.

[0080] It is of great advantage to the user of a laser manipulationsystem to be able to see at a glance what the effect of the laser willbe at positions remote from the actual laser focal point. Therefore, thederived isothermal contours are displayed on a computer screen. In anisotropic medium the contours are rings. The ring diameter depends onthe laser power and the laser duration. As the laser power and the laserduration are changed by the user, the rings expand or contractaccordingly. This enables the user to see exactly what the potential forembryo damage is with the laser, and to adjust the parametersaccordingly.

[0081] In application to the drilling of ZP, it is a further advantageof the invention that the size of the hole to be drilled is representedwell by the diameter of the 140° C. peak-temperature isotherm. Thus, forany conditions of laser power and laser pulse duration, the diameter ofthe T_(max)=140° C. isotherm gives the diameter of the hole that will bedrilled in the ZP.

[0082] An example of the invention is shown in the FIG. 7, in which theisotherms are shown superimposed on a scale image of an oocyteindicating the peak thermal excursion that will be experienced atdifferent distances from the laser beam focus by different parts of theoocyte.

[0083] The present invention is further illustrated by the followingexamples which should not be construed as further limiting. Numerousmodifications and alternative embodiments of the present invention willbe apparent to those skilled in the art in view of the foregoingdescription. Accordingly, this description is to be construed asillustrative only and is for the purpose of teaching those skilled inthe art the best mode for carrying out the present invention. Details ofthe structure may vary substantially without departing from the spiritof the invention, and exclusive use of all modifications that comewithin the scope of the appended claims is reserved. Such equivalentsare intended to be encompassed by the following Examples and claims.

EXAMPLES Example 1 Laser Module, Microscope Specifications, and Design

[0084] A Nikon TE-300 laser-equipped inverted microscope was used, whichhas the IR laser beneath the stage. Laser light proceeds up through theobjective and is focused on the target. The laser module (ZLTS, HamiltonThorne Research, Beverly, Mass.) has maximum power 200 mW at λ=1480 nm,and is utilized in pulsed mode. Peak transmitted laser power through theobjective is approximately 140 mW. Pulse duration is adjustable in halfmillisecond increments from 0.5 to 25 ms. In addition to the laser diodeitself, the laser module includes control board, adjustable collimatinglens and dichroic mirror, and is inter-locked to prevent potentiallyhazardous operation.

[0085] The laser module fits into the microscope filter cube slot underthe stage and the beam is deflected by a dichroic mirror along the opticaxis of the microscope. The nearly collimated beam is directed towardthe back aperture of a 40×, 0.60 numerical aperture (NA) objective lens,designed to maximize transmission at 1480 nm. The long working distance(WD=3 mm) objective lens is used to focus the laser beam on to thetarget specimen; it has high transmission ratio in the infrared range(typically about 71% at 1480 nm). The collimation of the beam isadjusted to give parfocality of IR and visible light. Biologicalspecimens, e g., bovine oocytes or mouse pre-embryos, are maintained inan aqueous culture medium in clear polystyrene Petrie culture dishes(Falcon, Lincoln Park, N.J.). The incident focused laser beam travelssequentially through air, plastic, and then the aqueous solution beforereaching the target area of the zona pellucida (ZP).

[0086] Delivered laser energy at the objective was measured using athermal detector head (Ophir Optronics, Peabody, Mass.) placedimmediately above the stage with objective removed. Transmission of theobjective was determined by placing an identical opposed objective abovethe focal point to recollimate the beam which passed into the detector.Using the derived transmission, 71% at λ=1480 nm, maximum beam power atthe focal point was estimated at 103 mW, allowing for reflection at thepolystyrene Petrie dish and absorption through 75 μm H₂O. Pulse durationwas checked using an oscilloscope (Tektronix TDS 360) monitoring laserradiation scattered on to a Ge photodiode detector (B1918-01,Hammamatsu, Bridgewater, N.J.). In a preliminary measurement, the focalbeam radius in air was determined by intercepting the beam at the focalpoint with a knife edge which was moved across the beam at known ratewhile the IR intensity was monitored with the Ge detector. At the focus,the radius to the 1/e² intensity level was R=3.1±0.5 μm. The focal conehalf angle in the medium is 26.7°.

Example 2 Laser-Assisted Zona Dissection with Bovine Eggs

[0087] Bovine eggs are readily available. They constitute a useful andcost effective model for testing laser-assisted zona drilling protocols.The Nikon TE300 inverted microscope is used with illumination source andcondenser above specimen and objective below. The Petrie dish specimenchamber (wall thickness 1 mm, diameter 50 mm) is on a motorized stage.The eggs settle at the bottom of the dish and the objective is focusedon the edge of the ZP, about 75 μm into the medium.

[0088] A typical zona drilling is shown in FIG. 8 for a bovine oocyte.The egg diameter is about 150 μm and the thickness of the zonapellucida, the translucent shell surrounding the egg, is about 12 μm.The laser power was set to 100 mW delivered at the focal point and threepoints in the ZP corresponding to pulse duration 1.5, 3, and 4.5 ms.Beam diameter to 1/e² was 6.2 μm, independent of beam power. The holediameters are 4.3, 7.8, and 10.5 μm; the hole diameter depends more onpulse duration than on laser beam size.

[0089] In FIG. 9A an electron photomicrograph of several channels in abovine egg is shown. Repeated firing at constant laser pulse length 25ms and laser power 50 mW produced channels of consistent size.

[0090] The close-up in FIG. 9B emphasizes the sharply drilled wall ofeach channel. The channels have clear cylindrical cross sections ofwidth approximately 12 μm with no significant beam broadening at eitherend. The channels reflect a thermal dissolution “melting” temperatureand its locus is not coterminous with the beam edge. Although noevidence of thermal damage can be seen surrounding the target area, thetransient thermal excursion may cause damage invisible under themicroscope to nearby living cells. The thermal history of the laserdrilling is derived as described below.

Example 3 Thermal Predictions

[0091] Standard Conditions

[0092] The objective (NA=0.6, 40×, infinity corrected) used transmits71% of the incident energy at λ=1480 nm. The typical laser beam powerarriving at the vicinity of the ZP following transmissive, reflective,and attenuation loss is calculated as 100 mW. The typical beam focalradius is calculated as a=3 μm, assuming the beam is passing up throughan inverted microscope into medium of refractive index n=1.333. Thethermal history or isothermal contour expected along the path of thelaser beam and especially in the vicinity of the pre-embryo is derivedas described below.

[0093] Thermal Constants and Source Function Geometry

[0094] Using medium comprised of almost pure water and the cellularmaterial is approximately 80% water, the infrared absorptances in themedium and cell at λ=1480 nm are approximately that of H₂O and cannot besignificantly lower. Similarly, the thermal conductivity of the materialwill not be higher than that of water. By taking the absorptance asidentical to that of H₂O, α=21 cm⁻¹, a lower limit to the temperatureproduced is obtained. On the time scales of interest (1-25 ms), the onlysignificant mode of heat loss from the heated liquid will be thermalconduction, since convection does not have time to develop and radiationmay be neglected. The thermal conductivity of water is 6×10⁻³ w/cm/° atroom temperature, but since the thermal excursion during the laser pulseis typically 100-200° C., we take the conductivity of the medium asK=6.8×10⁻³, corresponding to H₂O at 100° C.

[0095] The laser path is approximated as three regions: (1) a converging cone of semiangle θ; (2) the waist region of the beam; and (3) adiverging cone with semiangle θ. The beam has higher net power at theconverging than at the diverging cone, due to attenuation. The conesemiangle is θ=arcsin (NA/n) 26.7°. For the thermal calculation weapproximate the waist region as a cylinder of radius a=3 μm and length2α·cot θ.

[0096] The laser beam converges through the medium to a focal point 75μm above the floor of the Petrie dish. Absorption will only be importantin the medium and may be neglected in the Petrie dish. Self-focusing ofthe IR beam will not significantly affect the thermal distributionoutside the beam and is ignored. The radiation intensity is symmetric inazimuthal coordinate, varying in intensity only with axial distance andradius. Ignoring angular variation, the heat diffusion equation inaxisymmetric cylindrical coordinates may be written Equation  1:${\frac{\partial T}{\partial t} = {{k\frac{\partial^{2}T}{\partial r^{2}}} + {k\frac{1}{r}\frac{\partial T}{\partial r}} + {k\frac{\partial^{2}T}{\partial z^{2}}} + \frac{S}{\rho \quad C_{p}}}},$

[0097] where the heat diffusion coefficient is k=K/ρC_(p), andρC_(p)=4.18 j/cm³, with ρ the liquid density and C_(p) its specificheat, so k=1.6×10⁻³ cm²/s. Variation of the diffusion coefficient withtemperature is small and is ignored. The origin is taken as the focalpoint, with r and z as the radial and axial distances. The sourcefunction S represents the laser heating power per unit volume and variesacross the entire domain.

[0098] The source functions in the three regions of the laser path arenormalized to the laser power P reaching the focal plane so that theconverging beam is more intense below the waist and attenuates as itprogresses upward, diverging above the waist. The central cylinderlength is short (2a.cot θ=12 μm), and attenuation is ignored in region2.

[0099] The source functions for attenuated uniform beam in the threeregions are Equation  2-Converging:$S_{1} = {\frac{\alpha \quad P}{2{\pi ( {1 - {\cos \quad \theta}} )}} \cdot \frac{1}{r^{2} + z^{2}} \cdot ^{{\alpha {({z^{2} + r^{2}})}}^{1/2}}}$Equation  3-Waist$S_{2} = \frac{\alpha \quad P}{\pi \quad a^{2}}$Equation  4-Diverging:$S_{3} = {\frac{\alpha \quad P}{2{\pi ( {1 - {\cos \quad \theta}} )}} \cdot \frac{1}{r^{2} + z^{2}} \cdot ^{- {\alpha {({z^{2} + r^{2}})}}^{1/2}}}$

[0100] The Gaussian profile is approximated by applying the factorAe^(−2((arctanr/z)/θ)) ² to S₁ and S₃, where A is a normalizationconstant, and for S₂, we use the factor Ae^(−2(r/α)) ² .

[0101] Boundaries for Solution of the Heat Diffusion Equation

[0102] Equation (1) was solved by finite element analysis (FEA) usingthe codes COSMOS (Structural Research Co., Los Angeles, Calif.) andMathlab PDE Toolbox (Mathworks Inc., Natick, Mass.) with sourcefunctions from Equations (2)-(4). The beam travels parallel to the axisof a cylinder of radius 100 μm and length 200 μm. For short pulses (τ<6ms) these dimensions are sufficient to make edge effects negligiblesince the thermal diffusion distance in time τ is x<30 μm. The axis is aNeumann boundary with zero radial temperature gradient. Since the beamis attenuated, the focal plane cannot be taken as a Neumann boundary.Therefore, the equation must be solved in the entire upper half plane.The cylinder end planes at z=±100 μm and cylinder wall at r=100 μm areboth Dirichlet boundaries with temperature held at T₀=37° C. The initialtemperature of the entire domain is taken as T₀.

[0103] Of practical interest is thermal history on the focal planeitself at various radial distances from the axis, which will giveinformation on the thermal excursion experienced by the nearestblastomeres.

[0104] Preliminary Verification

[0105] For comparison with an analytic case, the FEA was run with auniformly heated cylinder of radius 3 μm in a 200 μm-diameter rightcylinder containing H₂O at T₀=37, and with source function as inEquation (2), P=100 mW and α=21 cm⁻¹.

[0106] The analytic steady-state solution for the temperature differencebetween edge and center of an infinite cylindrical region of radiusR_(max) heated on axis by a uniform beam of radius R is${{{Equation}\quad 5}:{\Delta \quad T}} = {\frac{\alpha \quad P}{4\pi \quad K}\lbrack {1 + {2{\ln ( \frac{R_{\max}}{R} )}}} \rbrack}$

[0107] The FEA steady-state result was within 0.5% of the temperaturedifference from Equation (5). Therefore, the system is integratingcorrectly.

[0108] Standard Case

[0109] The initial temperature is taken in the medium at thephysiological T₀=37° C. in a cylinder of diameter 200 μm and length 200μm, with attenuated converging beam of NA=0.6. The field is divided into1.13×10⁵ elements. The same temperature T₀ is held at the end planes andat the radius R_(max)=100 μm. Beam power is 100 mW at the focal waist,with pulse duration 3 ms and radius 3 μm. The problem is solved in thehalf plane and has been converted to the full plane in FIGS. 10A and 10Bwhere the beam direction is bottom to top, shown for times 0.1 and 3 ms,respectively. At 0.1 ms the central temperature is 98.8° C. In FIG. 10B,at 3 ms the central temperature is 189° C. If the beam is flattopinstead of Gaussian, central temperature is reduced approximately 15%.In either case, these high temperatures imply the potential presence ofsuperheated medium. The temperature change from the initial value at anytime depends on the pulse duration and is directly proportional to thebeam power (see Equations (1)-(5)). Predictions for any beam power maybe scaled from the results reported.

[0110] The effect of attenuation is apparent in the slightly nonverticalslope of the isotherms as the beam progresses from below to above thewaist in FIGS. 10A and 10B. Apart from this effect, the converging anddiverging parts of the beam away from the end planes result in almostcylindrical isotherms. For the present purpose the focal plane is theonly region of interest in questions of cell heating. Therefore, thequestion of whether the focal plane temperature heating can beapproximated by a simpler (and faster) one-dimensional solution isexamined.

[0111] To test the effect of geometry the FEA system was run for theGaussian beam in the following three cases, all with power 100 mW, pulse3 ms, and radius 3 μm:

[0112] 1. Attenuated beam, NA=0.6 (converging cone, central cylinderradius 3 μm, converging/diverging cone);

[0113] 2. Unattenuated beam, NA=0.6 (same as above, exponential terms inEquations (2) and (4) set to unity); and

[0114] 3. Unattenuated beam, NA=0 (uniform central cylinder radius 3 μm,exponential terms set to unity).

[0115] The latter case corresponds to a constant cylindrical beam andhas one-dimensional symmetry, whereas the first two require twodimensions.

[0116] For all three cases, predicted temperatures on the focal planefor radii 5≦radius≦50 μm as function of time are within 0.5° C. over 10ms integration time. At least on the focal plane, in the present casethe converging and diverging beam produces heating very similar to thatfrom a long heated cylinder, and if the power is normalized to its valueat the focal plane, the effect of beam attenuation may be ignored.Hence, the mathematically simpler case of the cylinder can be used forpredicting the temperature history at points on the focal plane.Accordingly, the one-dimensional Gaussian elimination solution has beenused as set forth below.

Example 4 Solution in Cylindrical Symmetry

[0117] Gaussian Elimination

[0118] The FEA solution shows that radial temperature history is almostcylindrical with isotherms close to parallel to the axis over the regionof interest. If the heating effect of the beam is approximated as acylindrical region of constant radius “a” equal to its focal radius,Equation (1) can be written as a one-dimensional difference equation,which can be rapidly solved by Gaussian elimination^(17, 18) to give thethermal time history in the region near the laser focus. The advantageof this is that solution is much faster than FEA, and the case of purecylindrical geometry without converging and diverging beams gives a goodapproximation to the focal plane temperature. A Gaussian elimination(GE) code to model the thermal behavior in the radial dimension isdescribed herein.

[0119] The area 0≦r≦R_(max) is covered and the R_(max)=100 μm is chosenbecause at the laser pulse lengths considered, the thermal excursiondoes not reach R_(max) before the end of the pulse. The cylindrical areais divided into N shells, each separated by Δr=R_(max)/N. Accurateresults are obtained with Δr=100 μm. As boundary conditions, the initialtemperature and the temperature at r=R_(max) are set to T₀=37° C. Thetemperature gradient at beam center is set to zero. In the GE analysis,it is assumed that the beam is flattop rather than Gaussian: thisreduces central temperature by about 15%, but does not affecttemperature history outside the beam. Equation (5) is used to check theresult and find agreement within 0.1% between the GE code and Equation(5) for very long (200 ms) pulses, so the GE code gives correct resultsfor the analytic case.

[0120] Thermal Histories on Focal Plane

[0121] The GE solutions for temperature at various radial positions onthe focal plane are shown as function of time in FIG. 11. The standardcase of beam power 100 mW, pulse duration 3 ms, in a 200 μm rightcylinder initially at 37° C. is used. At beam center the peaktemperature reaches over 170° C. Temperature falls off sharply as thepulse terminates due to the short diffusion time over 3 μm. As expected,the thermal excursion seen near the beam has approximately the sameduration as the laser pulse.

[0122] Superheating

[0123] The calculated (GE) peak central temperatures at typical beampowers with beam radius 3 μm in water are given for flattop beam focalspots of various pulse durations in Table 1 with steady-state valuesfrom Equation (5) for reference. Again, the region of the analysis istaken as a cylinder of length 200 μm and radius 100 μm. The calculatedtemperature of water exposed to the beam is very high, with peak centralbeam temperature ranges up to above 200° C., corresponding to highlysuper-heated water. The question is whether this temperature is real orwhether a phase change occurs which would reduce the central temperatureand form a column of vapor bubbles along the beam axis near the focalpoint. Miotello and Kelly¹⁹ have examined the formation rate ofhomogeneous nuclei and subsequent explosive phase change in superheatedliquids. In the absence of a surface for heterogeneous nucleation,homogeneous nucleation is necessary for rapid phase change, and its ratedepends strongly on how close the liquid is to the critical temperature(T_(C)=647 K for H₂O). The homogeneous nucleation rate becomessignificant near 300° C. However, at and below 250° C., the rate ofhomogeneous nuclei formation in water is negligible and, in the absenceof sites for heterogeneous nucleation, boiling within a 25 ms pulse isnot expected. If nucleation sites are present in the liquid remote fromthe walls, boiling could occur. While it is possible that the ZP orlocal suspended particles may provide sites for nucleation, it isunlikely since sharp solid edges are generally more favorable.

[0124] Thermal calculations indicate that highly superheated water isbriefly formed in the focal waist of the IR beam. Heat is conductedmainly radially away from the waist, and regions near the beam will beheated by conduction. Superheated water is an excellent solvent, and itis not surprising that the region of ZP within the focal waist rapidlydisappears.

[0125] After the beam is turned off any vapor bubbles formed would beexpected to collapse in less than 1 ms, releasing heat to the liquid. Ifa significant degree of phase change had occurred, the temperature atthe beam core would be held to close to 100° C., which would result inlower radial temperatures in the focal plane. The presence ofsuperheating could be tested by measuring local temperature excursions.TABLE 1 Peak Central Temperatures Pulse Power (mW) Duration (ms) At EndPulse (° C.) Steady State (° C.) 100 1.5 155.4 233.9 100 3.0 172.3 233.9100 4.5 182.3 233.9 100 6 189.3 233.9 100 10 201.9 233.9 100 15.0 211.7233.9 100 25 223.0 233.9 80 15.0 176.8 194.5 40 15.0 106.9 115.8

Example 5 Temperature Check from Probe Beam Steering

[0126] Beam Steering

[0127] In order to confirm the predicted temperature excursion, thechange in local refractive index around the IR focal waist caused by thetemperature excursion was estimated. The steering effect caused by thethermal-induced refractive index gradient on an orthogonal probe beamwas used. Estimates show that a 2° change in ray direction would bepossible for light passing near the IR heated water column.

[0128] Optical System

[0129] The setup is shown in FIG. 12. The IR beam passes vertically intoa 1×1 mm glass-walled rectangular tube (Vitrocom, Mountain View, N.J.)containing distilled H₂O. The laser is set to pulse at 4 Hz, while beampower and pulse duration are varied. The IR beam focus is set atapproximately 200 μm above the bottom wall of the rectangular tube. TheIR beam focal diameter has already been measured at close to 2a=6 μm. Ahorizontal probe continuous wave (cw) HeNe laser beam is focused with anf=120 mm lens into the region of the IR beam focus, with the HeNe opticaxis orthogonal to the IR beam axis. The HeNe probe beam radius to the1/e² points at its focal point was measured by knife intercept as22.3±1.2 μm. The probe covers the region of interest near the IR focus.The HeNe beam is then focused with a cylindrical lens on to a 1 mmaperture in front of a Si detector. By scanning the aperture+detector inthe direction orthogonal to both laser axes, the angle by which theprobe beam is refracted (or scattered) by the thermal field around theIR beam is determined. The system is used to measure the maximum anglethrough which the probe beam is turned at given pulse length/power. Thisis then compared with the angle derived from the predicted thermalfiled.

[0130] Probe Ray Steering

[0131] The curvature of the probe ray is proportional to (and in thedirection of) the normalized refractive index gradient ∇ n/n. In the IRbeam focal plane the temperature field, and consequently the refractiveindex, in the specimen must be radially symmetric around the IR axis.The refractive index will decrease toward the IR axis during the IRpulse, and a probe ray traveling in the focal plane of the IR beam willbe steered away from the axis (see FIG. 13). The steering angle ψ in acentrally symmetric refractive field²⁰ is given by the expressionEquation  6$\Psi = {\pi - {2 \cdot {\int_{R}^{\infty}{\lbrack {r\sqrt{( \frac{nr}{b} )^{2} - 1}} \rbrack^{- 1} \cdot {r}}}}}$

[0132] where b is the impact factor, r is radial distance from the IRbeam axis, n(r) is the refractive index, and R=b/n(R) is the distance ofclosest approach.

[0133] The refractive index n(T) of superheated water is given byIAPWS²¹ from 0 to 150° C. This covers the required range except for thebeam center. For higher temperatures, the IAPWS data was extrapolated.Using the GE solutions for the thermal field to calculate n(r), theexpected probe ray steering by the changed refractive index during thelaser pulse as a function of IR laser power and pulse length wasderived. The impact factor covers the range 0<b<22 μm due to the largeprobe beam diameter.

[0134] The maximum beam bending is a measure of the maximum temperaturegradient, which occurs adjacent to the beam radius and corresponds tothe specific temperature field. The predicted values for pulse duration25 and 2.5 ms are shown and maximum measured steering angles are givenin FIG. 14 for IR power 20-100 mW and pulse duration 1-25 ms. The laserpower shown in FIG. 14 is that delivered at the focal point, allowingfor absorption after 200 μm H₂O.

[0135] After the beam is turned off, any vapor bubbles formed would beexpected to collapse in less than 1 ms, releasing heat to the liquid.Possible phase change is neglected in the time scales of interest.Consequently, the predictions derived from Equation (1) to computetemperatures in the beam focal plane can be used.

[0136] For higher laser delivered power, the central temperature risesand the thermal gradient becomes steeper. The predicted values thereforeincrease. The measured values show a tendency to reach a limiting valueat high laser powers in the region where the predicted temperatureis >200° C., implying that the gradient has maximized. This is unlikelyto reflect phase transition: once phase change nucleates at thesesuper-heated temperatures, it would be expected to continue until themedium reaches T=100.

[0137] If the medium can reach a maximum temperature of only 100° C. dueto phase change, the predicted refractive index gradient never riseshigh enough to cause the observed beam bending, but flattens out atθ=0.6°. Since the observed maximum ray bending angle is θ=1.8°, theresults are consistent with central beam temperature >100° C.

[0138] “Melting Point” of Zona Pellucida

[0139] The diameter of the channels cut in the ZP of three bovineoocytes have been measured by pulses of power 100 mW and duration 1.5-25ms, using a 3 μm radius beam under standard conditions. The channels cutin the ZP of murine two-cell pre-embryos have also been measured by thesame beam. The channel diameter versus pulse duration is shown for bothspecies in FIG. 15. The GE model is applied to determine temperaturehistory on the focal plane at various distances from the beam axis andthe peak temperature calculated at each position is shown in FIG. 15.

[0140] The bovine ZP material appears to undergo dissolution (melt)between 140 and 150° C., the temperature decreasing with longer pulsetime. The murine ZP appears to be more easily removed and vanishes atabout 5-10° C. lower temperature than the bovine.

[0141] Descloux and Delacretaz²² measured the thermal dissolution ofmouse ZP at temperatures 61-73° C. over times up to 1000 s, and deriveda rate constant. This measurement is extrapolated to estimate the mouseZP dissolution temperature for 10 ms pulse as 111° C., lower than theexperimental estimate averaged over the ZP from mouse pre-embryos. Theminimum temperature that must be attained in the beam center in order toablate human ZP material in the pulse times used is likely to be between130 and 150° C.

Example 6 Conditions for Minimizing Collateral Damage

[0142] It is assumed that the beam central temperature should bemaintained at or above about 150° C. in order to penetrate the ZP withinpulse time of 10 ms. At the same time, the temperature at the surface ofthe nearest cell should be minimized. The typical distance to nearestcell is taken as 20 μm and use the thermal model above to derivetemperatures on the focal plane at 20 μm from the beam axis: in FIG. 16a graph is shown of the peak temperature reached during the pulse asfunction of pulse duration for various beam powers. In the present case,a typical thermal excursion would last on the order of the pulseduration and have peak amplitude shown. The cells, therefore, undergorapid heating and cooling of amplitude tens of degrees in a fewmilliseconds.

[0143] If thermal damage scales are assumed as a chemical reaction, thendamage would be proportional to τ·exp(−ΔQ/RT), where τ is pulseduration, T is the mean pulse temperature, and ΔQ is reaction energy.This would imply that the pulse time must be kept extremely short if thetemperature rise is significant.

[0144] Thermal excursion amplitude and duration should be minimized,therefore, pulse time is low. Short pulse duration and high beam powergive the most efficient system for maximizing beam cutting power andminimizing local heating. FIG. 11 indicates limiting lines correspondingto beam center peak temperatures of 150 and 110° C. The peak centraltemperature should exceed the limiting lines for rapid ZP ablation,while maintaining minimum peak temperature at 20 μm. It is evident thatthe laser pulse duration should be as short as possible, consistent witheffective ZP dissolution, i.e., preferably below 5 ms.

REFERENCES

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[0146] 2. W. B. Schoolcraft, T. Schlenker, G. S. Jones, and H. W. Jones,“In vitro fertilization in women age 40 and older: The impact ofassisted hatching,” J. Assist Reprod Genet. 12:581-584 (1995).

[0147] 3. M. Gennons, D. Nocera, A. Senn, K. Rink, G. Delacretaz,Pedrazzini, and J. P. Hornung, “Improved fertilization and implantationrates after non-touch zona pellucida microdrilling of mouse oocytes witha 1.48 μm diode laser beam,” Hum. Reprod 11:1043-1048 (1996).

[0148] 4. L. Gianaroli, M. C. Magli, A. P. Ferraretti, A. Fiorentino, J.Garrisi, and S. Munne, “Preimplantation genetic diagnosis increases theimplantation rate in human in vitro fertilization by avoiding thetransfer of chromosomally abnormal embryos,” Fertil. Steril.68:1128-1131 (1997).

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We claim:
 1. An optical injection system for use in conjunction with amicroscope having a turret supporting an objective having an opticalpath, the optical injection system comprising: a dichroic mirrordisposed between the turret and the objective, along the optical path; acollimating lens having an optical path directed to intersect thedichroic mirror; a laser source positioned to project a laser beamthrough the collimating lens and along the collimating lens opticalpath.
 2. The system of claim 1, wherein the laser source comprises aninternal laser powered from an electronic package.
 3. The system ofclaim 1, wherein the laser source further comprises fiber optic cableproviding optical energy from a laser.
 4. The system of claim 3, whereinthe laser is one of an internal laser and an external laser.
 5. Thesystem of claim 1, wherein the laser source operates at a wavelength (λ)of about 1480 nm.
 6. The system of claim 1, wherein the microscopecomprises one of an inverted and a non-inverted microscope.
 7. Thesystem of claim 1, wherein the objective comprises a shortened objectiveto provide a standard 45 mm parfocal unit module.
 8. The system of claim1, wherein the turret mounts on a removable turret adapter, the turretadapter being one of a plurality of turret adapter designs, therebycreating a universal mounting system, which enables the opticalinjection system to be mounted to the microscope.
 9. The system of claim8, wherein the turret adapter enables a method of orienting the lasersystem in any rotational position on a turret objective port.
 10. Thesystem of claim 1, further comprising an electronic package disposedseparate from the system.
 11. The system of claim 1, further comprisingan electronic package built-in to a board in a computing apparatus. 12.The system of claim 11, wherein the laser is coupled to a fiber andmounted inside the electronic package, and a second end of the fiber ismounted in the optical injection system in place of the laser.
 13. Thesystem of claim 1, wherein the system is suitable for the intended useof ablating, dissecting, moving, holding, or otherwise effectingbiological cells or tissue with a laser source.
 14. The system of claim1, wherein the laser beam is confocal with an optical image.
 15. Thesystem of claim 1, wherein the laser source and collimating lens furthercomprise fiber optic cable with integrated collimating lens providingoptical energy from a laser.
 16. A method for calculating and displayingthe isothermal contours generated by a laser in a sample, the methodcomprising: applying a laser beam to the focal point of a sample,dividing the region near the focal point into cylinders coaxial with thebeam, deriving the maximum temperature reached during the laser pulse ofat least three points at arbitrary distances from the focal point,plotting the temperatures calculated as a function of distance from thefocal point sufficient to generate isothermal contours, and generating acomputer display of said isothermal contours corresponding to thetemperature calculations.
 17. The method of claim 16, wherein the sampleis placed in an isotropic medium and the isothermal contours aredisplayed as rings centered around the focal point.
 18. The method ofclaim 16, wherein a picture of the sample is displayed with theisothermal contours.
 19. The method of claim 16, wherein the sample isthe ZP of a pre-embryonic or embryonic cell.
 20. The method of claim 16,wherein the temperature at the focal point is 140° C.
 21. The method ofclaim 16, where the isotherm rings are displayed as a color or grayscalegraphic overlay on top of a live camera or video image.
 22. The methodof claim 21, wherein the calculation and real time display of isothermrings may be seen on a computer monitor and where changes in thecorresponding laser power and laser pulse time selected in theapplication program result in corresponding changes visible on themonitor.